Plant cells have rigid cell walls that determine the size, form and stability of the plant cell. These cell walls are comprised primarily of polymers of simple sugar monomers linked in a variety of linear or branched polymers known as polysaccharides. The most abundant simple sugar monomer is glucose, and the most abundant polymer is cellulose. Cellulose is a linear, unbranched polymer, comprised of β-1,4 linked glucose monomers. Other polysaccharides found in plant cell walls include hemicelluloses, which comprise a group of polysaccharides composed of β-1,4 linked glucose monomers having side chains which may include sugars other than glucose, including xylose, fucose, arabinose, and galactose. Hemicelluloses are a heterogeneous mixture of polysaccharides, the composition of which varies substantially for different plants. Hemicelluloses are defined, operationally, as that polymer fraction which may be extracted from the cell wall with alkali.
Pectins are another type of polysaccharide found in plant cell walls. Pectins are acidic polysaccharides, which are generally comprised primarily of galacturonic acid and rhamnose sugar monomers. Amylose, another common plant polysaccharide, is not a major component of cell walls, but instead acts primarily as a storage material for glucose, rather than as a structural polymer. Because amylose is composed primarily of α-1,4-linked glucose monomers, it is considered to be a related polymer from a biochemical and physiological perspective. The molecular structure of the cell wall and its biogenesis during growth are not completely understood.
The alignment of cellulose microfibrils in the cell wall changes during development of the plant cell. In isodiametric meristematic cells, the fibrils are oriented randomly in the plane of the wall. During the transition to extension growth, an increasingly parallel orientation of the newly deposited fibrils is observed. Cells that grow predominantly in one direction generally have parallel fibrils oriented generally perpendicular to the direction of growth. Plant cells having thick cell walls, such as epidermal and xylem cells, often have a multilayered microfibril structure. These walls may have thin layers of parallel fibrils, the direction of which changes from layer to layer by a substantially constant angle.
An important difference between the cell walls of trees and herbaceous plants is that tree cell walls have more complex xylem layers. The xylem types in cell walls of trees vary depending on the age of the tree and the position of the xylem in the tree. For example, young trees (less that eight years old for pine) or upper parts of the tree (with fewer than 6-8 growth rings) produce so-called juvenile wood xylem. Older parts of the tree produce so-called late wood xylem. Xylem cells have additional cellulose-rich secondary wall layers incorporated into the primary wall, which may become thickened and develop an increased tensile strength and resistance to pressure. The secondary cell wall comprises three additional layers, namely the S1, S2 and S3 layers. In mature wood and late wood (wood formed in autumn) the S2 layers are thicker and the cellulose fibrils have higher angles (both of which are commercially desirable traits), when compared to juvenile or early wood.
The secondary walls may comprise a considerable amount of lignin in addition to cellulose, pectins and hemicelluloses. The S1 layer is generally highly lignified, the S2 layer is lightly lignified, whereas the S3 layer is also highly lignified. Lignin is an insoluble polymer that is primarily responsible for the rigidity of plant stems. Specifically, lignin serves as a matrix around the polysaccharide components of some plant cell walls. In general, the higher the lignin content, the more rigid the plant. For example, tree species synthesize large quantities of lignin, with lignin constituting between 20% to 30% of the dry weight of wood. The lignin content of grasses ranges from 2-8% of dry weight and changes during the growing season. In addition to providing rigidity, lignin aids in water transport within plants by rendering cell walls hydrophobic and water impermeable. Lignin also plays a role in disease resistance of plants by impeding the penetration and propagation of pathogenic agents.
The presence and composition of lignin in plant cell walls is desirable for some applications and undesirable for others. In forestry trees, lignification reduces access by chemicals during pulping or during timber treatment. Similarly, in forage crops, the lignification reduces the digestibility of the forage crops for animals. Lignin is, however, an essential component of cell walls and provides structural support for the plant. Two major goals for the forestry industry are reduced rotation times and reduced costs of extracting pulp from wood. To reduce rotation times, young trees need to have enhanced growth characteristics, and have the wood characteristics of older trees. To reduce the costs of extracting pulp from wood, young trees need to have a reduced or modified lignin content. Similarly, for forage crops, an objective is to increase the digestibility and efficiency of the crop without adversely altering its growth and structural properties. By reducing lignin content of cereal stubble the time required to degrade the stubble in the soil will be greatly reduced. Furthermore by reducing the lignin content of high biomass producing cereals such as maize or sorghum the ability to utilize biomass for conversion to ethanol will be greatly enhanced.
Forage digestibility is affected by both lignin composition and concentration. Lignin is largely responsible for the digestibility, or lack thereof, of forage crops, with small increases in plant lignin content resulting in relatively high decreases (>10%) in digestibility (Buxton and Russell, Crop Sci. 28:538-558, 1988). For example, crops with reduced lignin content provide more efficient forage for cattle, with the yield of milk and meat being higher relative to the amount of forage crop consumed. During normal plant growth, an increase in the maturity of the plant stem is accompanied by a corresponding increase in lignin content and composition that causes a decrease in digestibility. This change in lignin composition is to one of increasing S:G ratio (syringyl/guaiacyl units). When deciding on the optimum time to harvest forage crops, farmers must therefore choose between a high yield of less digestible material and a lower yield of more digestible material.
As discussed in detail below, lignin is formed by polymerization of different monolignols that are synthesized in a multistep pathway, each step in the pathway being catalyzed by a different enzyme. It has been shown that manipulation of the number of copies of genes encoding certain enzymes, such as cinnamyl alcohol dehydrogenase (CAD) and caffeic acid 3-O-methyltransferase (COMT) results in modification of the amount of lignin produced; see, for example, U.S. Pat. No. 5,451,514 and PCT Publication No. WO 94/23044. Furthermore, it has been shown that antisense expression of sequences encoding CAD in poplar leads to the production of lignin having a modified composition (Grand et al., Planta (Berl.) 163:232-237, 1985). Quantitative and qualitative modifications in plant lignin content are known to be induced by external factors such as light stimulation, low calcium levels and mechanical stress. Synthesis of new types of lignins, sometimes in tissues not normally lignified, can also be induced by infection with pathogens.
Lignin is formed by polymerization of at least three different monolignols, primarily para-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol. While these three types of lignin subunits are well known, it is likely that slightly different variants of these subunits may be involved in the lignin biosynthetic pathway in various plants. For example, studies suggest that both free monolignols and monolignol-p-coumarate esters may be substrates for lignin formation in grasses. The relative concentration of the monolignol residues in lignin varies among different plant species and within species. The composition of lignin may also vary among different tissues within a specific plant. The three monolignols are derived from phenylalanine or tyrosine in a multistep process and are believed to be polymerized into lignin by a free radical mechanism.
Coniferyl alcohol, para-coumaryl alcohol and sinapyl alcohol are synthesized by similar pathways. The first step in the lignin biosynthetic pathway is the deamination of phenylalanine or tyrosine by phenylalanine ammonia-lyase (PAL) or tyrosine ammonia-lyase (TAL), respectively. In maize, the PAL enzyme also has TAL activity (Rosier et al., Plant Physiol. 113: 175-179, 1997). The product of TAL activity on tyrosine is p-coumarate. The product of PAL activity on phenylalanine is trans-cinnamic acid which is then hydroxylated by cinnamate 4-hydroxylase (C4H) to form p-coumarate. p-Coumarate is believed to be hydroxylated by coumarate 3-hydroxylase (C3H) to give caffeate. The newly added hydroxyl group is then methylated by caffeic acid O-methyl transferase (COMT) to give ferulate. More recently, a caffeoyl-CoA O-methyl transferase (CAMT) enzyme has been hypothesized to play a role in the lignin biosynthetic pathway (Ye et al., Plant Physiol. 108:459-467, 1995).
Ferulate is conjugated to coenzyme A by 4-coumarate:CoA ligase (4CL) to form feruloyl-CoA. Reduction of feruloyl-CoA to coniferaldehyde is catalyzed by cinnamoyl-CoA reductase (CCR). Coniferaldehyde is further reduced by the action of cinnamyl alcohol dehydrogenase (CAD) to give coniferyl alcohol which is then converted into its glucosylated form for export from the cytoplasm to the cell wall by coniferol glucosyl transferase (CGT). Following export, the de-glucosylated form of coniferyl alcohol is obtained by the action of coniferin beta-glucosidase (CBG). Finally, polymerization of the three monolignols to provide lignin is catalyzed by phenolase (PNL), laccase (LAC) and peroxidase (PER). The formation of sinapyl alcohol involves an additional enzyme, ferulate-5-hydroxylase (F5H). For a more detailed review of the lignin biosynthetic pathway, see Whetton R and Sederoff R, The Plant Cell, 7:1001-1013, 1995 and Whetten R et al., Annu. Rev. Plant Physiol. Plant Mol. Biol. 49:585-609, 1998.
Cellulose Synthesis
The major source of dietary fiber for grazing animals comes from plant cell walls. Mammals possess no enzymes capable for breaking down the polysaccharides in plant cell walls. Instead animals such as ruminants depend on microbial breakdown of plant cell walls through fermentation in either the rumen or large intestine.
Fiber in plants is measured using the Neutral Detergent Fiber (NDF) technique in which plant samples are boiled in a solution containing sodium lauryl sulfate (van Soest in “Nutritional Ecology of the Ruminant”. Cornell University Press, Ithaca, N.Y., 1994). This detergent extracts water-soluble components such as sugars, lipids and organic acids. The remaining insoluble residue (fiber) is termed NDF and consists predominantly of plant cell wall components such as cellulose, hemicellulose, and lignin. The amount of cellulose and lignin in cell walls can be determined using the Acid Detergent Fiber (ADF) method where plant samples are boiled in sulfuric acid and sodium lauryl sulfate. The difference between NDF and ADF for a plant sample is normally considered to be the amount of hemicellulose (van Soest in “Nutritional Ecology of the Ruminant”. Cornell University Press, Ithaca, N.Y., 1994).
Stems of most forage species have greater NDF content than leaves. For example, for a temperate C3 grass in mid-flowering such as tall fescue (Festuca arundinacea), NDF content of leaves and stems is 50 and 70%, respectively (Buxton and Redfearn, J. Nutrition 127:S814-S818, 1997). In contrast, for a C4 tropical grass such as switchgrass (Panicum virgatum L.) the NDF content of leaves and stems is 70 and 85%, respectively. The digestibility of a forage is determined by cell wall content, so that legumes are more digestible than grasses because they contain less NDF. The NDF of a legume, however, is generally less digestible than that of grasses because a higher proportion of the NDF is made up by lignin. The increase of lignin as a component of NDF is also responsible for the decrease in digestibility of grasses at the time of flowering. In fact, ruminants can digest only 40-50% of NDF in legumes compared to 60-70% for grass NDF (Buxton and Redfearn, J. Nutrition 127:S814-S818, 1997). Digestibility of cellulose by ruminants is therefore directly related to the extent of lignification. Generally hemicellulose is more digestible than cellulose.
Cellulose is the most abundant carbohydrate in forage making up to 20-40% of dry matter (van Soest in “Nutritional Ecology of the Ruminant”. Cornell University Press, Ithaca, N.Y., 1994). The cellulose in forages consists predominantly of β1-4 glucan (85%) and smaller amounts of pentosans (e.g. xylose and arabinose; 15%). There appear to be two pools of cellulose within the plant cell wall, the difference being one is lignified and the other is not (van Soest in “Nutritional Ecology of the Ruminant”. Cornell University Press, Ithaca, N.Y., 1994). The lignified cellulose is mostly found in the primary cell wall and in the S1 outer layer of the secondary cell wall. Independent of lignification, it appears that cellulose possesses variability in nutritive quality (van Soest in “Nutritional Ecology of the Ruminant”. Cornell University Press, Ithaca, N.Y., 1994). This indicates that it is possible to alter the rate of cellulose digestibility by modifying the chemical composition of cellulose. This could be achieved through manipulating the actions of the cellulose synthesis and cellulose synthesis-like enzymes found in plant cells. One method to increase digestibility in this way is to increase the activity of the cellulose synthesis and cellulose synthesis-like enzymes responsible for synthesizing hemicellulose or to down regulate the cellulose synthesis and cellulose synthesis-like enzymes making cellulose. Hemicellulose is much more digestible than cellulose and is less likely to become lignified. Another way of manipulating cell wall composition is through modifying the rate and supply of primary components required for cellulose synthesis, i.e. of β1-4 glucan and pentosans such as xylose and arabinose. One way to achieve this is to modify the actions of soluble sucrose synthase and UDP glucose pyrophosphorylase enzymes that produce the UDP-glucose required for cellulose synthesis. This may be further modified by manipulating the actions of the large and small subunits of ADP-glucose pyrophosphorylase, the two enzymes that are rate-limiting steps in starch synthesis (Smith et al., Ann. Rev. Plant Phys. Plant Mol. Biol. 48:67-87, 1997).
Cellulose synthases are found in all tissues and cell types of plants and are involved in both primary and secondary cell wall biosynthesis. Cellulose synthase (cel or cesA) is a glycosyltransferase that utilizes UDP-glucose as a substrate in the polymerization of glucose residues to form 1,4-β-D-glucans (Richmond, Genome Biology I: reviews 3001.1-3001.6, 2000), thereby catalyzing the synthesis of cellulose (an aggregate of β-1,4-linked glucose residues as unbranched polymers). The CesA protein contains putative transmembrane domains and is thought to span the plasma membrane, where this catalytic component may interact with other proteins to form a cellulose synthase ‘complex’. In plants, cesA proteins are encoded by a multi-gene family, of which ten have been identified from Arabidopsis thaliana, nine from maize (Zea Mays) and eight from rice (Oryza sativa) (Arioli et al., Science 279:717-720, 1998; Holland et al., Plant Physiol. 123:1313-1324, 2000). Differential expression of the Arabidopsis CesA genes suggests these genes have different functions within the plant.
In addition to the cellulose synthase genes described above, plants have a superfamily of cellulose synthase-like (CSL) genes, whose amino acid sequences are related to the CesA genes (Richmond and Somerville, Plant Physiol. 124:495-498, 2000; Richmond and Somerville, Plant Mol. Biol. 47: 131-143, 2001; Hazen et al., Plant Physiol. 128: 336-340, 2002). The CSL proteins are predicted to be integral membrane proteins and contain a highly conserved motif that is characteristics of glycosyl transferases. This family of proteins synthesizes repeating β-glycosyl subunits and the CSLs may be involved in the biosynthesis of plant cell wall components, for example hemicellulose. Using sequence data, the CSL superfamily can be divided into several distinct families, for example Arabidopsis has six CSL families (with 40+ members), whereas maize lacks one of these families but has a further two families.
Manipulating expression of genes in the cel/CSL superfamily would alter the chemical composition of plant cell walls in forage plants. Altering cell wall biosynthesis therefore provides an opportunity to increase digestibility of the plant dry matter. This may be achieved by increasing the amount of carbon in the plant allocated to cellulose biosynthesis at the expense of lignin biosynthesis. Alternatively, decreasing the amount of cellulose biosynthesis and increasing the amount of water-soluble carbohydrates would have a similar effect. Furthermore, specifically increasing hemicellulose levels in the plant cell walls at expense of cellulose would also increase forage digestibility. By utilizing specific promoters in combination with the cel and CSL genes it is possible to increase or decrease cellulose and hemicellulose levels in the leaf or stem.